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dsRNA carrier for interfering bx-cpl-2 gene expression and application thereof in control and research on bursaphelenchus xylophilus

A technology of bx-cpl-2 and interference carrier, which is applied in the field of plant disease and insect pest control, and can solve problems such as unsustainable interference, stable interference, and instability

Inactive Publication Date: 2013-08-14
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the means of RNAi has also become an important method for the gene function of pine wood nematode, but the soaking method is used at present, and the dsRNA synthesized in vitro is soaked in pine wood nematode to analyze the function of the gene. This method has great shortcomings, mainly It is shown that the interference cannot be sustained, and it is extremely unstable. How to realize the stable interference of RNA is also a further goal of the present invention

Method used

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  • dsRNA carrier for interfering bx-cpl-2 gene expression and application thereof in control and research on bursaphelenchus xylophilus
  • dsRNA carrier for interfering bx-cpl-2 gene expression and application thereof in control and research on bursaphelenchus xylophilus
  • dsRNA carrier for interfering bx-cpl-2 gene expression and application thereof in control and research on bursaphelenchus xylophilus

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Experimental program
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Effect test

Embodiment 1

[0085] Example 1: Construction of Agrobacterium-mediated filamentous fungus expression dsRNA vector—PHD-RH

[0086] Agrobacterium-mediated filamentous fungal transformation vector PHD-T, protoplasts transformed filamentous fungal RNAi vector Psilent-1 ( figure 2 ) is a known carrier, donated by researcher Liu Xingzhong, Institute of Microbiology, Chinese Academy of Sciences.

[0087] The construction of vector PHD-RH is mainly based on Agrobacterium-mediated transformation of filamentous fungi pDHt / hph As a backbone, its T-DNA region was modified, and a DNA sequence (Seq ID No.1) was synthesized by Shanghai Bioengineering Company. Its structure was KpnⅠ restriction site, Aspergillus nidulans tryptophan promoter, Multiple cloning site, intron, 3' multiple cloning site, Aspergillus nidulans tryptophan terminator, BstxⅠ restriction site (5'------3'). Insert the synthetic sequence into the vector pDHt / hph Between the KpnⅠ and BstxⅠ sites, the positive clones were identified ...

Embodiment 2

[0090] The cultivation and separation of embodiment 2 pine xylophilus

[0091] 1. Cultivation of B. xylophilus

[0092] B. xylophilus was cultured with Botrytis cinerea grown on PDA medium.

[0093] Sterilize the PDA medium at 121°C for 20 minutes by high-pressure steam, and pour it into a petri dish at about 60°C; after the medium is cooled, insert Botrytis cinerea into the ultra-clean bench and place it in a constant temperature incubator at 25°C for cultivation; 3 to After 4 days, pine wood nematodes were sterilized with hydrogen peroxide and inserted into a petri dish, and placed in a constant temperature incubator at 25°C for cultivation; after 6 to 7 days, the petri dish was taken out of the incubator and stored at 4°C.

[0094] 2. Isolation of B. xylophilus

[0095] Wrap the medium containing pine xylophilus with four layers of gauze, suspend it in a beaker filled with sterilized distilled water; place the beaker in a constant temperature incubator at 25°C for 24 hour...

Embodiment 3

[0130] Embodiment 3. Construction of RNA interference vector

[0131] The pGM-T carrying the sense strands of Bx-cpl-1 and Bx-cpl-2 genes and the PHDT-RH vector were double-digested with HindⅢ and XbaⅠ enzymes, respectively, and the Bx-cpl-1 and Bx-cpl The sense chain pGM-T vector of the -2 gene was digested with a fragment of about 350 bp, and the fragment size was the same as expected. Recover the target fragment and digested vector PHDT-RH for ligation, transformation, and screening of positive transformants. In the positive transformants, the downstream of the sense strand of the target gene has introduced IT (Intron) sequence. Sent to Beijing Sangong for sequencing. The PHDT-RH vector inserted into the positive sense strand and the PHDT-RH vector of the antisense strand of the target gene after correct sequencing were double digested with SpeI and BglII enzymes. The target fragment and the digested vector are recovered, ligated, transformed, screened, and sequenced. Th...

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Abstract

The invention discloses a dsRNA carrier for interfering Bx-cpl-2 gene expression and an application thereof in control and research on bursaphelenchus xylophilus, and belongs to the plant disease and insect control technology. The dsRNA interference carrier characterized in that a complete or partial sense strand of the Bx-cpl-2 gene and an antisense strand complementary to the sense strand are forwardly inserted at a polyclone locus of a skeleton carrier in sequence. The dsRNA carrier can be used for controlling and researching the bursaphelenchus xylophilus.

Description

technical field [0001] The invention belongs to the technical field of plant disease and pest control, and in particular relates to a dsRNA carrier that interferes with the expression of Bx-cpl-1 gene and its application in the control of pine wood nematode. Background technique [0002] Pine wood nematode (Bursaphelenchus xylophilu) is a parasitic nematode that is extremely harmful to forests, and it mainly harms coniferous trees represented by pine species. In the twentieth century, pine trees in Japan were devastated by pine xylophilus infection. In my country, pine wood nematode was first discovered in Nanjing in 1982. After that, the epidemic spread rapidly, causing great damage to my country's pine forest resources, and its economic losses are incalculable. Pine wood nematode disease has even directly threatened my country's Zhangjiajie and other world natural and cultural heritage landscapes. [0003] Pine wood nematode disease is known as "the cancer of pine trees"...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12R1/645
Inventor 陈国华王殿东白婷成新跃梅眉谢丙炎
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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